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AlphaLISA SureFire Ultra Human and Mouse Total IRF8 Detection Kit, 500 Assay Points
AlphaLISA SureFire Ultra Human and Mouse Total IRF8 Detection Kit, 500 Assay Points
AlphaLISA Surefire Ultra Total Protein
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total IRF8 assay is a sandwich immunoassay for quantitative detection of total IRF8 in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Protocol Time | 2h at RT |
| Sample Volume | 10 µL |
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total IRF8 assay is a sandwich immunoassay for quantitative detection of total IRF8 in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 Assay Points
Part #:
ALSU-TIRF8-A-HV
List price
USD 722.00
Your online price:
Unit Size: 500 Assay Points
Part #:
ALSU-TIRF8-A500
List price
USD 2,441.00
Your online price:
Unit Size: 10,000 Assay Points
Part #:
ALSU-TIRF8-A10K
List price
USD 14,688.00
Your online price:
Unit Size: 50,000 Assay Points
Part #:
ALSU-TIRF8-A50K
List price
USD 46,690.00
Your online price:
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
AlphaLISA SureFire Ultra Human and Mouse Total IRF8 Detection Kit, 500 Assay Points
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Ultra Human and Mouse Total IRF8 Detection Kit, 500 Assay Points
Product information
Overview
Interferon Regulatory Factor 8 (IRF8) is a transcription factor that plays essential roles in myeloid cell development, dendritic cell differentiation, and immune responses to intracellular pathogens. IRF8 forms heterodimeric complexes with PU.1 to regulate myeloid lineage commitment, promoting monocyte and dendritic cell development. It is critical for development of classical and plasmacytoid dendritic cells and regulates expression of IL-12 and type I interferons. Loss-of-function mutations in IRF8 cause severe immunodeficiency with absent dendritic cells. IRF8 functions as a tumor suppressor in chronic myeloid leukemia and other myeloid malignancies.
The AlphaLISA SureFire Ultra Human and Mouse Total IRF8 is a sandwich immunoassay for the quantitative detection of total IRF8 in cellular lysates, using Alpha Technology.
Formats:
- The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
- The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
- The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
- The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
- Cell and tissue lysates
- Antibody modulators
- Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
- Cellular kinase assays
- Receptor activation studies
- High-throughput screening for preclinical studies
How it works
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted protein. AlphaLISA assays require two bead types: Acceptor and Donor beads. Acceptor beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted protein, the two antibodies bring the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.
Assay validation
BRD4 PROTAC-mediated downregulation of IRF8 expression in THP-1 cells
THP-1 cells were seeded in a 96-well U-bottom plate (200,000 cells/well) in complete medium. Cells were treated with increasing concentrations of BET PROTAC, ARV-771 for 18 hours in complete medium. After treatment, cells were washed with HBSS and lysed in Lysis Buffer.
IRF8, BRD4 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra kits. For the detection step, 10 µL of cell lysate (approximately 2,000 cells for IRF8, BRD4 and 20,000 for ERK1/2) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, degradation of BRD4 lead to IRF8 downregulation in THP-1 cells (a model cell line for acute-myeloid leukemia).
BRD4 PROTAC-mediated downregulation of IRF8 expression in THP-1 cells
THP-1 cells were seeded in a 96-well U-bottom plate (200,000 cells/well) in complete medium. Cells were treated with increasing concentrations of BET PROTAC, MZ-1 for 18 hours in complete medium. After treatment, cells were washed with HBSS and lysed in Lysis Buffer.
IRF8, BRD4 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra kits. For the detection step, 10 µL of cell lysate (approximately 2,000 cells for IRF8, BRD4 and 20,000 for ERK1/2) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, degradation of BRD4 lead to IRF8 downregulation in THP-1 cells.
Assay versatility
Differential IRF8 expression in various cell lines
Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO2 and were lysed with Lysis Buffer at a density of 0.5 x 106 cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 106 cells/mL.
IRF8 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Total IRF8 protein is highly expressed in B lymphoma cell lines like RPMI 8226 and Karpas 299, with no expression detected in Jurkat or A431 cells.
Assay sensitivity
Assay sensitivity - cell lysate
Cell lysate was prepared from THP-1 cells lysed at a density of 1.5 x 106 cells/mL in Lysis Buffer.
Lysates were serially diluted in Lysis Buffer and IRF8 levels were assayed using the AlphaLISA SureFire Ultra kit. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. The assay can detect Total IRF8 down to 200 cells.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Ultra
|
| Detection Modality |
Alpha
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Sample Volume |
10 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
IRF8
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Inflammation
|
| Unit Size |
500 Assay Points
|
Resources
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